Project 1: Mothes ? Conformational events underlying HIV-1 envelope antagonism SUMMARY The HIV-1 envelope glycoprotein (Env) trimer is the sole viral protein exposed on the virus surface and as such represents a main target for entry inhibitors and vaccines to treat HIV-1/AIDS. A major barrier in targeting HIV- 1 Env has been the conformational flexibility that conceals functional centers. We have developed single- molecule Frster Resonance Energy Transfer (smFRET) methods to directly visualize the conformational states and structural dynamics of HIV-1 Envs in the context of the native trimer on the surface of HIV virions. Our results revealed that the functional Env trimer exists primarily in a closed conformation (State 1), but has inherent access to the open CD4-bound conformation (State 3) through one necessary asymmetric trimer intermediate (State 2). When we asked how the existing high-resolution structures relate to the states observed by smFRET, we discovered that they all correspond to either State 2 or 3. The all-important structure of State 1 of HIV-1 Env, which is the target of the majority of broadly neutralizing antibodies, remains unknown. Going forward, an understanding of the structure of State 1 will be critical as this is the conformation that most small molecule inhibitors will first engage. To this end, we will use smFRET to guide the field towards experimental conditions that permit a structural characterization of State 1. Defining conformational states of native Env trimers will also provide a mechanistic framework for how small molecule inhibitors developed by this Program antagonize Env. Conformational blockers specifically bind to State 1 and prevent the adoption of the CD4-bound conformation. In contrast, CD4 mimics shift the conformational landscape towards the more open State 2 and 3 conformations. Applied to new compounds development by this Program Project, smFRET will continue to reveal conformation selectivity, determine conformational trajectories and lead to mechanistic insights into irreversible Env inactivation. Understanding the molecular mechanisms by which compounds inhibit, prematurely activate or drive HIV-1 envelope off-pathway will guide the rational design of new classes of inhibitors.